SUMMARY: Since its cloning in 1995 and its identification (under this grant) as an unprecedented intramembrane aspartyl protease in 1999, Presenilin has been implicated in a remarkable array of signaling events in all metazoans. PS was discovered through research on Alzheimer?s disease, but it was soon shown to confer functions necessary for life, including as the protease that enables Notch nuclear signaling. Therefore, continuing to decipher the structure, functions, and protein and small-molecule regulators of PS is a priority for fundamental cell biology. At the same time, the invariant cerebral accumulation of amyloid b-protein (Ab) decades prior to symptoms has made PS/g-secretase a rational target for mechanistic and therapeutic study in AD. Despite its pleiotropic role in biology, the protease?s structure was only recently reported at 3.4, leaving many structure-function details unresolved, and small molecules that can safely and potently modulate its cleavage of APP are just entering human trials. For these reasons, three collaborators with deep experience in the study of Presenilin over 20 years wish to apply a range of methods in cell biology, biochemistry, cellular imaging, genetics, stem cell biology and medicinal chemistry to tackle some of the thorniest questions in PS/g- secretase biology. To wit, based on our new model of PS processivity (Bolduc et al., eLife 2016), can we insert many different FAD-causing PSI mutations into the protein and identify which residues contribute to the S1?- S2?-S3? active-site pockets we recently found to mediate the tri-peptide cleavages? What is the biological mechanism of coordinated b- and g-secretase processing within a novel complex? How does a new PS protein interaction discovered here, namely with GLT-1, help modulate glutamate uptake by astrocytes and perhaps also change g-secretase function? Can one identify and validate GSMs that are sufficiently potent yet selective to chronically shift g-secretase cleavages from toxic Ab42/43 to protective Ab37/38 peptides? How can we learn more about the participation of PS/g-secretase in stem cell development in the CNS? Here, we propose numerous interrelated aims that incorporate three cross-cutting themes that unite our work. First, all 3 projects will build on a new explanatory mechanism of g-secretase processing discovered under this grant: that its processive tri-peptide cleavages are dictated by 3 pockets in the presenilin enzyme controlling the engagement of substrates with the catalytic site. Second is our strongly shared interest in small-molecule modulators of the PS/g-secretase complex. Each of our 3 projects includes aims that will examine GSMs to assess effects on the PS functions we are individually studying. A third cross-cutting theme comes from our shared use of a large library of PS1 and APP plasmids, sensitive ELISAs, and advanced microscopy reagents -- all developed under this grant. In sharing these approaches and reagents, our PPG is not a collection of marginally related aims but rather a highly integrated program in which we use variations on common themes & methods to address major unsolved questions about g-secretase, the RIP mechanism, and its safe modulation to treat and prevent AD.